The average American consumes about 450 mg of cholesterol per day and produces an additional 500 to 1000 mg in the liver and other tissues. Although cholesterol is essential to health, excess serum cholesterol has been implicated in atherosclerosis, heart attack and stroke, and is a leading cause of death in the United States, accounting for approximately 600,000 deaths per year.
Although mammals can endogenously synthesize fats, including cholesterol, their main source is direct absorption from the diet. The liver is able to partially regulate the levels of circulating lipids by modulating the rates of fatty acid uptake, esterification into triglycerides or oxidation, processes that are coordinated at the transcriptional level by a small number of nuclear receptors.
Serum cholesterol levels are also regulated by the rate of transport of this lipid out of the cells into the blood, a process that is mediated with the help of protein carriers called lipoproteins. Two important classes of these protein carriers are low-density lipoproteins (LDL) and high-density lipoproteins (HDL). LDL is responsible for transporting cholesterol from the liver to various tissues and body cells while HDL is largely responsible for transporting excess or unused cholesterol back to the liver where it may be metabolized to bile acids for excretion.
Healthy cholesterol levels for LDL should be lower than 130 mg/dl while HDL should be more than 50 mg/dl. When the body has too much LDL, i.e., above 160 mg/dl, cholesterol starts to accumulate along the interior walls of arteries leading to a buildup of fatty deposits in the coronary arteries and other blood vessels leading to atherosclerosis and atherosclerotic cardiovascular diseases.
Because the diet of most western societies is rich in animal products, the ability to be able to modulate serum cholesterol concentrations in vivo independently of the diet would be particularly useful for preventing coronary heart disease and other disorders associated with the high dietary intake of fat. Accordingly the development of agonists, antagonists, inverse agonists, partial agonists and antagonists, as well as pan agonists and antagonists, for the nuclear receptors involved in regulating the transcription of proteins involved in lipid metabolism and transport would have immediate application in treating disorders associated with alterations in fat metabolism, transport or uptake.
Such nuclear receptors include the peroxisome proliferator activated receptors (PPARα, β/δ and γ) the farnesoid receptor (FXR), the Pregnane X-Receptor (PXR), Constitutive Androstane Receptor (CAR) and the liver X receptors (LXRα and LXRβ). The various alternate names, and representative GenBank Accession numbers for these receptors are shown below.
AlternativeAccessionReceptor Name and SubtypeNamesNo.PPARα (Peroxisome ProliferatorPPARα,NM_005036Activated Receptor-αNR1C1PPARβ(Peroxisome Proliferator ActivatedPPAR-βXM_004285Receptor)PPAR-δ,NR1C2NUC1, FAARPPARγ, Peroxisome Proliferator ActivatedPPARγ,XM_003059Receptor-γNR1C3LXR-β, (Liver X receptor-βLXR-β, UR,U07132NR1H2NER-1,RIP15, OR1LXR-α, (Liver X receptor-α)LXRA, XR2,U22662NR1H3RLD1
AccessionReceptor Name and SubtypeAlternative NamesNo.FXR (Farnesyl X receptor)FXR, RIP14, HRR1NM_005123NR1H4PXR (Pregnane X-Receptor)PXR.1, PXR.2, SXR,NM_0038892 IsoformsONR1, xOR6, BXRNM_022002NR1I2AF364606CAR α (Constitutive AndrostaneCAR1, MB67XM_042458Receptor)NR1I3CAR β(Constitutive AndrostanemCAR1 (mouse)Receptor)NR1I4
These receptors bind to hormone response elements as heterodimers with a common partner, the retinoid X receptors (RXRs) (see, e.g., Levin et al., Nature (1992), Vol. 355, pp. 359-361 and Heyman et al., Cell (1992), Vol. 68, pp. 397-406). The table below lists such RXR receptors.
Receptor Name and SubtypeAlternative NamesAccession No.RXRα, (Retinoid X-Receptor-α)RXRαNM_002957NR2B1RXRβ(Retinoid X-Receptor-β)RXRβ, H2RIIBPXM_042579NR2B2RXRγ (Retinoid X-Receptor-γ)RXRγXM_053680NR2B3
The three proteins encoded by the RXR genes are all able to heterodimerize with any of the receptors above, and these heterodimers can be activated by both RXR ligands (i.e., rexinoids) as well as ligands for the partner nuclear receptor.
Role in Lipid Metabolism
Although all the nuclear receptors above play a role in controlling overall lipid metabolism, distinct classes of receptor play defined cell type specific roles in the entire process.
The peroxisome proliferator-activated receptors (PPARs) for example, are fatty acid and eicosanoid inducible nuclear receptors, that are regulated by fatty acid derivatives. The three PPAR isoforms have distinct patterns of expression and function within the body.
PPARα is mostly expressed in brown adipose tissue, liver, kidney, duodenum, heart and skeletal muscle. PPARγ expression, by contrast, is mainly found in brown and white adipose tissues and, to a lesser extent, in the large intestine, the retina and in some parts of the immune system. PPARβ is the most ubiquitously expressed isotype and is found in higher amounts than α and γ in almost all tissues examined, except the adipose tissue.
PPARα participates in the control of fatty acid transport and uptake by stimulating the transcription of genes encoding the fatty acid transport protein (FATP), the fatty acid translocase (FAT/CD36) and the liver cytosolic fatty acid binding protein (L-FABP). The metabolism of triglyceride-rich lipoproteins is modulated by PPARα dependent stimulation of the lipoprotein lipase gene, which facilitates the release of fatty acids from lipoprotein particles, and by the down regulation of apolipoprotein C-III. Furthermore, PPAR α upregulates apolipoproteins A-I and A-II in humans, which leads to an increase in plasma high-density lipoprotein (HDL) cholesterol. Additional PPAR α target genes participate in the mitochondrial fatty acid metabolism, in ketogenesis and in microsomal fatty acid ω-hydroxylation by cytochrome P450 ω-hydroxylases that belong to the CYP4A family.
By comparison, PPARγ plays a major role in regulating adipose tissue differentiation and fat storage, which is a major site for the overall control of lipid homeostasis in the body.
The Farnesoid X Receptor (FXR) is an orphan receptor initially identified from a rat liver cDNA library (Forman, B M, et al., Cell 81: 81 687-693 (1995)) that plays a major role in the homeostasis of cholesterol in the body. FXR is most abundantly expressed in the liver, intestine, kidney and adrenal, and is activated by several naturally occurring bile acids including chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), and the taurine and glycine conjugates of these bile acids.
It is now known that FXR functions as a bile acid sensor that participates in the regulation of cholesterol homeostasis by controlling the conversion of cholesterol to bile acids. High bile acid levels suppress the conversion of cholesterol to bile acids by activating FXR that acts to suppress the expression of the cholesterol 7 α-hydrolase gene (Cyp7A) and other enzymes involved in bile acid synthesis. Cyp7A is responsible for the first enzymatic step in the conversion of cholesterol to bile acids and represents the key rate limiting enzymatic step in bile acid synthesis. Cyp7A belongs to the Cytochrome P-450 family of enzymes, and is found exclusively in the liver. FXR is also involved in controlling the synthesis of isoprenoid derivatives (including cholesterol). In the ileum, FXR mediates the expression of the intestinal bile acid binding protein (IBABP) that is involved in the cellular uptake and trafficking of bile acids.
High cholesterol levels also lead to the accumulation of oxidized derivatives of cholesterol, such as 24(S), 25-epoxycholesterol, 22(R)-hydroxycholesterol, and 24(S)-hydroxycholesterol which are activators of the Liver X Receptors (LXRs).
These compounds tend to accumulate in the cell under conditions of elevated cholesterol in the cell and act on LXR to coordinate an increase in the transcription of genes involved in cholesterol transport out of the cell, the synthesis of enzymes involved in the metabolic conversion of cholesterol to bile acids, and an increase in the expression of genes involved in fatty acid synthesis. By promoting the metabolic conversion of cholesterol to bile acids, these LXR agonists also promote the transfer of cholesterol from the periphery to the liver for catabolism and excretion.
In mammals two forms of LXR exist (α and β) with different patterns of expression. LXR α is expressed predominantly in the liver, with lower levels found in kidney, intestine, spleen and adrenal tissue (see, e.g., Willy, et al. (1995) Gene Dev. 9(9):1033-1045), while LXR β is ubiquitously expressed.
The LXRs are also regulated by fatty acids, and these metabolites have opposing (antagonistic) effects on LXR transcriptional activity. Thus LXR antagonists, including fatty acids and their derivatives, act to decrease cholesterol transport out of the cell, decrease fatty acid synthesis and the conversion of cholesterol to bile acids by acting to suppress the transcription of genes involved in these pathways.
The target genes regulated by LXR, which effect these changes, are important enzymes involved in sterol metabolism, transport and metabolic diseases. Genes involved in sterol transport for example, including the ATP binding cassette transporters ABCA1, ABCG1, ABCG5 and ABCG8 as well as the cholesterol transport protein apolipoprotein apoE (a component of LDL), have been shown to have direct links to various disease syndromes.
Mutations in sterol transporter ABCA1 give rise to Tangier disease, and result in an almost complete absence of HDL cholesterol and promote accumulation of cholesterol within peripheral tissues. Both the ABCG5 and ABCG8 genes are both linked to human genetic syndromes including sitosterolemia, characterized by perturbed cholesterol transport.
As a component of all lipoprotein fractions, ApoE plays an important role in cholesterol transport. In ApoE knock out mice, the animals rapidly develop hypercholesterolemia and atherosclerosis, even when kept on a low fat diet. In man, mutations in apoE are associated hyperlipidemia and rapid onset of atherosclerosis (see, Atherosclerosis (1995), Vol. 112, pp.19-28).
LXRs also regulate fatty acid metabolism by controlling expression of the sterol response element binding protein 1c (SREBP1c), the master transcriptional regulator of fatty acid synthesis, and the enzymes that participate in this metabolic pathway. The ability of LXRs to regulate these enzymes has important consequences for carbohydrate and lipid homeostasis throughout the body.
The regulation of cholesterol transport, metabolism and SREBP1c expression suggests that LXR modulators (either alone, or in combination) have the potential to be useful in the treatment of diseases associated with defects in cholesterol transport, fatty acid metabolism and cholesterol metabolism.
Thus, there is a need for compounds, compositions and methods of selectively modulating the activity of nuclear receptors, including LXRs, FXR, PPARs, PXRs, CARs and orphan nuclear receptors for use in the treatment, prevention, or amelioration of one or more symptoms of numerous disease states.